The present invention relates to the genomic sequence of the human xcexc-opioid receptor gene and its variants, polymorphisms and mutations and their use.
As is known, the human xcexc-opioid receptor controls pain perception, xe2x80x98rewardxe2x80x99 mechanisms and further important physiological functions. It is the highly specific target for morphine, the classical painkiller in contemporary medicine. In addition, it is target for further medically important analgesics, anesthetics and therapeutics such as e.g. methadone and fentanyl and commonly used addictive substances such as e.g. heroin and methadone. A number of (patho)physiological, biochemical, pharmacological findings, and observations from xcexc-opioid receptor knock out mice demonstrate that the xcexc opioid receptor gene is playing a major role in analgesia and anesthesia, and in the development and maintenance of addictions (dependence on opiates, alcoholism and other forms of dependence). Therefore, variants in the regulating, coding and intronic regions of this gene may contribute to genetic risk for addictions. In addition, such variants may affect the responsiveness of this receptor to endogenous and exogenous receptor ligands.
Addictions are common diseases of international dimension and cause, in general, scarcely comprehensible grave economic damages to the amount of billions up to trillions, not to speak of the deleterious psychosocial consequences for the individual, his family and society.
The genomic sequence of the human xcexc opioid receptor gene is not known. So far only the cDNA of the xcexc opioid receptor gene has been described; the first cDNA of a xcexc opioid receptor was cloned by means of probes targeted against conserved regions of the xcex4 opioid receptor from a rat cDNA gene bank by Chen et al. (Molecular cloning and functional expression of a mu opioid receptor from rat brain. Mol. Pharmacol. 44, 8-12, 1993), the first human MOR cDNA by Wang et al. (Mu opiate receptor: cDNA cloning and expression. Proc. Natl. Acad. Sci. U.S.A. 90, 10230-10234, 1993). The only promoter sequence so far known was cloned from a mouse gene bank (Min. et al., Genomic structure and analysis of promoter sequence of a mouse xcexc opioid receptor gene. Proc. Natl. Acad. Sci. U.S.A. 91, 9081-9085,1994).
Actual findings obtained from xe2x80x98knockoutxe2x80x99 mice with an interrupted xcexc opioid receptor gene show clearly that the analgesic and xe2x80x98rewardxe2x80x99 inducing and dependence inducing effects of morphine are brought about specifically through the xcexc-opioid receptor subtype, yet not through xcex4- and xcexa opioid receptor subtypes. Accordingly, the xcexc opioid receptor is mandatory for morphine action in vivo (Matthes et al., Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the xcexc opioid receptor gene. Nature 383, 819-823, 1996). A series of pharmacological and other studies have shown that the xcexcopioid receptor expressed in brain is of key importance for the development of tolerance, addiction and analgesia (Reisine, Neurotransmitter Receptors V., Neuropharmacology 34, 463-472, 1995). In this context, endorphins come into consideration as endogenous ligands. Exogenous xcexc opioid receptor ligands such as morphine, codeine, methadone and fentanyl have been used for a long time as analgesics and therapeutics in clinics.
As is known, the clinical use of opiates causes undesired side effects such as breathing disturbances, miosis, nausea and vomiting, sedation, depression and dependence.
It is the objective of the invention to identify and provide the genomic sequence of the human xcexc opioid receptor which may be used as basis for the development of specific and efficient analgesics, anesthetics and therapeutics for addiction, in particular for developing e.g. analgesics not having addictive side effects, or for developing diagnostic kits.
According to the invention it was possible to identify and provide the genomic sequence of the human xcexc opioid receptor gene and of variants, polymorphisms and mutants in specific populations.
The genomic DNA sequence of the human xcexc opioid receptor amplified and sequenced according to the invention consists of
1) a promoter region (incl. 5xe2x80x2 regulatory region) SEQ ID no. 1 of a length of altogether 2412 bp.
The promoter is cloned according to methods known per se by amplifying the five various genomic DNA regions covering the human xcexc opioid receptor promoter region.
2) Intron 2 the genomic DNA sequence of which is between nucleotides 855 and 856 of the cDNA sequence (or between nucleotides 643 and 644 related to A of the translation starting point) of a length of 773 bp according to SEQ ID no. 2.
3) the 5xe2x80x2 region of intron 1 the genomic DNA sequence of which is behind nucleotide 502 of the cDNA sequence (or behind nucleotide 290 related to the translation starting point) of a length of 383 bp according to SEQ ID no. 3; and the 3xe2x80x2 region the genomic DNA sequence of which is in front of nucleotide 503 of the cDNA sequence (or in front of nucleotide 291 related to the translation starting point) of a length of 538 bp according to SEQ ID no. 4.
4) the 5xe2x80x2 region of intron 3 the genomic DNA sequence of which is behind nucleotide 1376 of the cDNA sequence (or behind nucleotide 1164 related to the translation starting point) of a length of 300 bp according to SEQ ID no. 5; and the 3xe2x80x2 region the genomic DNA sequence of which is in front of nucleotide 1377 of the cDNA sequence (or in front of nucleotide 1165 related to the translation starting point) of a length of 400 bP according to SEQ ID no. 6.
Sequencing of the intron is carried out analogously to sequencing of promoters.
In addition, there was detected that the human cDNA sequence already known consisting of 2162 bp is defective in the first 16 nucleotides. According to the invention the cDNA has SEQ ID no. 7.
The genomic sequence of the human xcexc opioid receptor gene is represented in illustrations 1a and 1b in a survey.
Four different transcription starting positions were identified in positions 212, 329, 371 and 421 bp upstream of the translation star site (ATG).
Variants, polymorphisms and mutants in specific populations are marked by base exchange. According to the invention these are base exchanges in up to 100 nucleotide positions, preferably up to 37 base exchanges are identified. In the cDNA region, in particular, preferably up to 20 base exchanges are identified, most preferably up to 11 base exchanges.
In a scheme according to the invention base exchanges take place in the following positions of the promoter (=RG=5xe2x80x2 regulatory region), of the exons (=cDNA sequence) and of the introns:
This exchange may be optionally effected only in one of the above-mentioned nucleotide positions, in any of the positions mentioned, or in all of the positions mentioned.
According to the invention such interindividual allelic variations in the coding and regulatory DNA regions of the human xcexc opioid receptor gene may be associated with individually different responsiveness to anesthetics/therapeutics and/or addictive substances, as well as an increased genetic risk for substance, dependence or e.g. physiologically modified pain sensitivity. Thus, they are used as a starting point for the development of individually tailored therapeutics, the prediction of individual therapeutic xe2x80x98responsexe2x80x99 and the genetic risk of addiction, contributing thus, at the same time, to prevention. In addition, they are the starting point for the genotypification of individuals to examine relevant environmental factors for the development of addition on the long term.
Thus, according to the invention the sequences serve the development of therapeutics, in particular analgesics/anesthetics and drug-therapeutics. They are used for building up genes and vectors which form the basis for the development of these pharmaceutically relevant substances.
Apart from that, diagnostic test kits are provided for predicting the risk of addiction such as e.g. dependence on opiates, alcoholism, dependence on cocaine etc. or for predicting the individual responsiveness to various analgesics and/or anesthetics and for the individually differing disposition to side effects of pharmaceutics.
In further perfecting the invention is was detected that associations of variants detected in the xcexc opioid receptor gene occur with diseases or clinically relevant phenotypes.
A significant association/specific connection of the Asnxe2x86x92Asp mutation in position 40 of the amino acid sequence (position 330 of the cDNA sequence) with family-related alcoholism was detected. This sequence position is not only connected with alcoholism as a specific form of addiction but also with a general disposition to addiction as it finds its expression in man by the nearly usual, very frequently occurring clinical form of multiple drug dependence (here caused by the simultaneous abuse of alcohol, opiates and cocaine). In addition, this mutation results in a functional state of the human xcexc opiate receptor which leads to a modified responsiveness of the receptor to ligands (endogenous and exogenous ligands including therapeutics, anesthetics and drugs) and a modified responsiveness of the receptor to a prolonged or repeated application of these ligands, thus being of importance to the development of tolerance (desensitization of the receptor to a chronic administration of pharmaceuticals) and dependence.
Furthermore, there was detected that the specific combinations of variants in the 5xe2x80x2 regulatory region are connected with a disposition to various diseases, in particular addictions. In particular, the positions xe2x88x921793/4Txe2x86x92A, xe2x88x921768ins22, xe2x88x921699insT, xe2x88x921469Txe2x86x92C, and xe2x88x921320Axe2x86x92G are of relevance in this respect. Especially the combination xe2x88x921793/4A, xe2x88x921768 wild type, xe2x88x921699insT, xe2x88x921469T and xe2x88x921320G is connected with a disposition to cocaine abuse and with a disposition to addiction in general (including alcoholism and dependence on opiates) and is functionally accompanied by a modified expression of the receptor. This combination described (xe2x80x98haplotypexe2x80x99) can better describe the real, overall functional state of the receptor in the pathophysiological situation than an individual, associated variant. This analysis is based on the concept that the differing functional (dysfunctional) receptor states are not exclusively based on individual mutations but that they are also caused by the individual xe2x80x9cpolymorphicxe2x80x9d total gene sequence (gene profile) as a unity determining the functions.
Accordingly, the object of the invention is a method for identifying the dispositions to diseases wherein the DNA of a proband is isolated and genotypied in selected positions and subsequently compared with the reference DNA sequence. Forms of execution where the positions xe2x88x921793/4Txe2x86x92A, xe2x88x921768ins22, xe2x88x921699insT, xe2x88x921469Txe2x86x92C and xe2x88x921320Axe2x86x92G are genotypied are preferred.
To detect the disposition to addiction it will be sufficient if 3 of these 5 positions will be investigated, yet it would be preferable to genotype all 5 positions to obtain reliable data. Additionally, it is possible to investigate position 330 of the cDNA sequence in connection with a disposition to alcohol by means of the test method.
Genotypification is effected by sequencing or other methods suited for detecting point mutations. They involve PCR-based genotyping methods such as e.g. allele-specific-PCR, other genotyping methods using oligonucleotides and methods using restriction enzymes.
The method according to the invention allows a. o. to detect a disposition to family-related alcoholism, addiction to cocaine and a dependence on opiates. Furthermore, an individually differing responsiveness to receptor agonists and antagonists may be detected.